The Role And Mechanism Of Deep Hypothermia Regulated Autophagy In Inflammation And Apoptosis Of PC12 Cells Following Oxygen Glucose Deprivation

Background:Deep hypothermia is usually used for circulatory arrest,such as aortic or complex congenital heart disease surgery of cardiovascular surgery and intracranial giant aneurysm or cerebrovascular malformation of neurosurgery,which is referred to as deep hypothermic circulatory arrest(DHCA)and introduced into surgical operations for sixty years.It can stop the circulation to provide 'bloodless' surgical field as well as protect the brain effectively via lowering the temperature to 15-20?.The relatively safe limitation period is 40-60 min.If the period is more,the probability of postoperative cerebral ischemic stroke and cognitive dysfunction will increase.Although the protective role of deep hypothermia for circulatory arrest has already been confirmed by some animal experiments and clinical practices,its protection mechanism has not yet fully understood and it has been the focus at home and abroad all the time.Autophagy is a progress of 'self-eating' which exists in eukaryotic cells.Intracellular double membrane structures combined with lysosomes to clear their own organelles and protein in order to maintain the cell homeostasis and eliminate supernumerary or damaged organelles.In some cases,autophagy inhibits apoptosis for cell survival,but autophagy can cause cell death sometimes.Autophagy is widely involved in various physiological and pathological processes which is the hot research topic in biomedical field.In recent years,some studies found that autophagy,apoptosis and inflammation play an important role in the nervous system diseases including ischemia-reperfusion injury,brain trauma and neural degenerative diseases and so on.How deep hypothermia impacts on autophagy in neurons,if there is influence,what effect does deep hypothermia regulated autophagy to the neurons,which is no research report.PC 12 cells are an ideal model for brain ischemia and other nervous system diseases in vitro,and the cells in vitro are uniform which are not affected by other cells.They are easy to control the environment factors and have good repeatability which is the base of life science research.So this topic using in vitro models to explore the role of deep hypothermia in oxygen glucose deprivation treated PC 12 cells and the effect on autophagy.And further study the role and mechanism of deep hypothermia regulated autophagy in inflammation as well as the role and mechanism of deep hypothermia regulated autophagy in apoptosis.From the three aspects,to discuss the role and mechanism of deep hypothermia in neuroprotection,to provide a new theoretical basis for deep hypothermia in neuroprotection,also to provide new targets and thoughts for deep hypothermia in brain protection.Part I The Role of Deep Hypothermia in Oxygen Glucose Deprivation Treated PC12 Cells and AutophagyObjective:Establishing oxygen glucose deprivation model with PC 12 cells to study the role and mechanism of deep hypothermia in it.To observe the effects of deep hypothermia on autophagy,and analyze the role of autophagy in oxygen glucose deprivation model accompanied with deep hypothermia.Methods:The PC 12 cells were cultured in high-glucose Dulbecco's Modified Eagle Medium(DMEIM)medium supplemented with 15%horse serum,2.5%fetal bovine serum,100 U/mL penicillin,and 100 g/mL streptomycin and were maintained in a humidified incubator with 5%C02 at 37?.Cells were digested by trypsin for passage and only cells showing in logarithmic phase was used in the study.Establishing oxygen glucose deprivation model:cells were cultured in DMEM without glucose and serum and put into a hypoxia chamber,saturated with a gas mixture of 1%O2,94%N2 and 5%CO2,for 1 h at 37?.Six experimental groups were assigned.Control group was maintained for the same period in normoxic conditions and the growth medium at 37?.DH group:PC 12 cells were maintained in a 18? incubator,normal oxygen and normal DMEM medium for 1 h.3-Methyladenine(3-MA)group:3-MA,the autophagy inhibitor,was added to the culture media at the concentration of 5 mmol/L ahead of 1 h.OGD group:the PC12 cells were in glucose-free DMEM and hypoxia for 1 h followed by reperfusion for 24 h.OGD + DH group:the PC 12 cells were cultured in serum-free and glucose-free medium into a hypoxia chamber for 1 h at 18?,with warming to a temperature of 37°C at a flow rate over 30 min.OGD + DH + 3-MA group:3-MA was used to treat PC 12 cells at the concentration of 5 mmol/L 1 h prior to OGD,then the cells underwent OGD and 18?.1.To observe the cellular morphological changes in each group by inverted microscope.2.To test cell viability with the CCK-8 assay in each group.3.To observe the number of apoptotic cells with Hoechst33258 staining assay by fluorescence microscope.4.Flow cytometry analysis of cell apoptosis using the Annexin V-FITC and PI apoptosis detection kit.5.Flow cytometry analysis of reactive oxygen species(ROS)using DCFH-DA probe.6.Flow cytometry analysis of Ca2+ using Fluo-3 AM probe.7.pmCherry-C1-EGFP-LC3B plasmid was amplified,withdrew and transient transfected into cells to observe the number of autophagosome and autolysosome by fluorescence microscope and analyze the autophagic flux.8.Observation of transmission electron microscope for autophagosome,autolysosome and cellular ultrastructure.All data are expressed as the means ? standard deviation(SD).One-way ANOVA(F test)was used to assess the difference among multiple groups followed by q test.Analysis was performed by SPSS Statistics 19.0.Results:Morphological observation:Cells in normal control group,DH group and 3-MA group were high density,spindle-shaped,large body,long neurodendrite and axons,and intertwined with each other.Cells in OGD group were low density,small body,retracted neurodendrite and axons.Cells in OGD + DH group were spindle-shaped,partly retracted neurodendrite and axons.Cells in OGD + DH +3-MA group were lower density than OGD + DH group,retracted neurodendrite and axons.2.Cell viability of CCK-8 assay:Cell viability was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the viability of OGD group was significantly reduced(P<0.05).Compared with OGD group and OGD + DH + 3-MA group,the viability of OGD +DH group was significantly increased(P<0.05).3.Hoechst 33258 staining assay:The number of apoptotic cell was no statistically significant difference among control group,DH group and 3-MA group(P>0.05)and few apoptotic cell was observed.Compared with control group,the number of apoptotic cell of OGD group was significantly increased(P<0.05)and nuclear condensation and fragmentation were observed.Compared with OGD group and OGD + DH + 3-MA group,the number of apoptotic cell of OGD + DH group was significantly reduced(P<0.05).4.Flow cytometry analysis of cell apoptosis assay using the Annexin V-FITC and PI:The apoptosis rate was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the apoptosis rate of OGD group was significantly increased(P<0.05).Compared with OGD group and OGD + DH + 3-MA group,the apoptosis rate of OGD + DH group was significantly reduced(P<0.05).5.Flow cytometry analysis of ROS:The level of ROS was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the level of ROS of OGD group was significantly increased(P<0.05).Compared with OGD group and OGD + DH ?3-MA group,the level of ROS of OGD + DH group was significantly reduced(P<0.05).6.Flow cytometry analysis of Ca2+:The level of Ca2+ was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the level of Ca2+ of OGD group was significantly increased(P<0.05).Compared with OGD group and OGD + DH + 3-MA group,the level of Ca2+ of OGD + DH group was significantly reduced(P<0.05).7.Observation of fluorescence microscope for autophagy:The number of autophagosome and autolysosome was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the number of autophagosome and autolysosome and autophagic flux in OGD group was significantly increased(P<0.05)indicating oxygen glucose deprivation could activate autophagy.Compared with OGD group and OGD + DH + 3-MA group,the number of autophagosome and autolysosome of OGD + DH group was significantly increased(P<0.05)indicating deep hypothermia could further increase autophagy.8.Observation of transmission electron microscope for autophagy:Normal morphology of nuclei,mitochondria,rough endoplasmic reticulum and cytoplasmic Golgi complexes were observed in control,DH group and 3-MA group.Autophagosomes were hardly found incontrol and 3-MA group,and can be occasionally found in DH group.There was no statistically significant difference among these three groups(P>0.05).Compared with control group,the number of autophagosome and autolysosome of OGD group was increased(P<0.05),while nuclear chromatin condensation and mitochondrial vacuolation were found.Compared with OGD group,the number of autophagosome and autolysosome of OGD + DH group was further increased(P<0.05),and normal mitochondria in the cytoplasm was observed.Compared with OGD + DH group,the number of autophagosome and autolysosome of OGD + DH + 3-MA group was significantly reduced(P<0.05),nuclearchromatin condensation and mitochondrial vacuolation were observed.Conclusions:Deep hypothermia(18?)could effectively inhibit apoptosis to protect oxygen glucose deprivation treated PC 12 cells and reduce the level of intracellular ROS and Ca2+.Oxygen glucose deprivation could activate autophagy in PC 12 cells which could be further increased by deep hypothermia.As autophagy was inhibited with 3-MA,the protective role of deep hypothermia would reduce indicating that autophagy played an important role in protection of deep hypothermia for oxygen glucose deprivation treated PC 12 cells,while the mechanism of protection of deep hypothermia may be through increasing autophagy activation.Part II The Mechanism of Deep Hypothermia Regulated Autophagy in Oxygen Glucose Deprivation Treated PC12 Cells via TLR4 Signaling PathwayObjective:To explore the mechanism of deep hypothermia regulated autophagy in oxygen glucose deprivation treated PC 12 cells via TLR4 signaling pathway and to determine marker protein of autophagy and TLR4 signaling pathway,such as LC3,Beclinl,TLR4,NF-?B,MyD88,IL-1?,IL-6 and TNF-?.Methods:Six experimental groups were assigned including control group,DH group,3-MA group,OGD group,OGD + DH group and OGD + DH + 3-MA group.Western blotting was used to detect these highly correlated proteins including LC3,Beclin1,TLR4,NF-?B and MyD88.ELISA was used to detect IL-1?,IL-6 and TNF-?.Results:Western blotting analysis of autophagy:the ratio of LC3?/LC3? and the level of Beclinl was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the ratio of LC3?/LC3?and the level of Beclinl in OGD group was significantly increased(P<0.05).Compared with OGD group and OGD + DH + 3-MA group,the ratio of LC3?/LC3?and the level of Beclinl of OGD + DH group was significantly increased(P<0.05).Western blotting and ELISA analysis of TLR4 signaling pathway:the level of TLR4,NF-?B,MyD88,IL-1?,IL-6 and TNF-? was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the level of TLR4,NF-?B,MyD88,IL-1?,IL-6 and TNF-? in OGD group was significantly increased(P<0.05).Compared with OGD group and OGD + DH +3-MA group,the level of TLR4,NF-?B,MyD88,IL-1?,IL-6 and TNF-? of OGD +DH group was significantly reduced(P<0.05).Conclusions:Neuroprotective effects of deep hypothermia were associated with suppressing TLR4 signaling pathway.The protective mechanism of deep hypothermia may be down-regulation of TLR4 signaling pathway via increasing autophagy.Part ? The Mechanism of Deep Hypothermia Regulated Autophagy in Oxygen Glucose Deprivation Treated PC12 Cells via Mitochondrial Apoptosis PathwayObjective:To explore the mechanism of deep hypothermia regulated autophagy in oxygen glucose deprivation treated PC 12 cells via mitochondrial apoptosis pathway and to determine marker protein of mitochondrial apoptosis pathway,such as Bcl-2,Bax,Cytochrome c,Caspase-3,Caspase-9 and cleaved PARP-1.Methods:Six experimental groups were assigned including control group,DH group,3-MA group,OGD group,OGD + DH group and OGD + DH + 3-MA group.Western blotting was used to detect these highly correlated proteins including Bcl-2,Bax,Cytochrome c,Caspase-3,Caspase-9 and cleaved PARP-1.Results:Western blotting analysis of mitochondrial apoptosis pathway:the level of Bcl-2,Bax,Cytochrome c,Caspase3,Caspase9 and cleaved PARP-1 was no statistically significant difference among control group,DH group and 3-MA group(P>0.05).Compared with control group,the level of Bcl-2,Bax,Cytochrome c,Caspase3,Caspase9 and cleaved PARP-1 in OGD group was significantly increased(P<0.05).Compared with OGD group and OGD + DH + 3-MA group,the level of Bcl-2,Bax,Cytochrome c,Caspase-3,Caspase-9 and cleaved PARP-1 of OGD + DH group was significantly reduced(P<0.05).Conclusions:Neuroprotective effects of deep hypothermia were associated with suppressing mitochondrial apoptosis pathway.Deep hypothermia down-regulated mitochondrial apoptosis pathway via increasing autophagy may be one of protective mechanisms to ischemic neuron.